1. Field of the Invention
The invention relates generally to recombinant DNA technology methods employing novel protease and heat shock protein deficient bacteria for the expression of polypeptides. Significantly improved yields of protease sensitive polypeptide products are obtainable employing the disclosed methods. Several novel protease-deficient mutants are disclosed, including a ptr mutant lacking Protease III and mutants with degP and rpoH deficiencies.
2. Description of Related Art
The expression of proteins in bacteria is by far the most widely used approach for the production of cloned gene products, many of which have important medical and commercial application. It is common to first obtain a polypeptide encoded by a cloned or mutated gene in E. coli, a readily available and well-characterized bacterial host cell. However, for various reasons, expressed bacterial polypeptide products may not be active. In some cases this may be due to incorrect folding or failure of essential post-translation modification in the host cell. Additionally, yields and/or recovery of the polypeptide product may be low. Alternate expression systems are therefore desirable to overcome these problems.
Bacterial cells are generally preferred for protein/polypeptide expression for several reasons. As a rule, prokaryotic cells are easier to grow than eukaryotic cells such as human cell lines. Additionally, a wealth of sophisticated molecular genetic tools and thousands of mutants with useful phenotypes are available to manipulate for particular expression problems.
The expression of proteins in bacteria benefits from the availability of a range of strong promoters capable of directing high levels of transcription of the desired heterologous gene. Translation of the mRNA is optimized by using a strong ribosome binding site (RBS) and substituting with the appropriate codons at the 3' of the gene. Such manipulations are routine and can result in very high rates of protein accumulation, often exceeding 50% of the total cell protein, provided that the protein is stable in vivo and is not susceptible to proteolytic degradation.
The expression of heterologous proteins in a form that is excreted from the bacteria into the extracellular fluid offers important technological advantages. First, once a protein has been exported from the cytoplasm, it becomes exposed to an oxidative environment which favors the formation of disulfide bonds, an important step in the correct folding of the polypeptide chain. In contrast, disulfide bond formation does not occur within the cytoplasm. Second, exported proteins are first synthesized with an N-terminal extension called the leader peptide which is precisely excised from the protein concomitant with transport through the membrane. As a result, export from the cytoplasm is an effective way of ensuring that the protein contains the correct N-terminal amino acid. On the other hand, proteins expressed in the cytoplasm often contain an additional methionine amino acid at their N-terminus. The presence of the additional N-terminal methionine is highly undesirable in pharmaceutical proteins where it is imperative that the protein sequence produced in bacteria is identical to the sequence of the corresponding protein in nature. Finally, it is desirable to express proteins in secreted form because of ease in separation from other cellular components.
A number of useful eukaryotic proteins have been cloned and expressed in bacterial cells, including human insulin and proinsulin, human and bovine somatotropins, interferons and tissue plasminogen activator. Recently, Huse and coworkers (1989) constructed a bacteriophage lambda system which allows the expression and rapid screening of mouse F.sub.ab antibody fragments in E. coli.
Escherichia coli has been the most widely used microorganism for the production of commercially important recombinant proteins. Despite the lack of certain kinds of post-translational processing and the production of endotoxins, E. coli presents numerous advantages for protein expression. Its genetics are well understood, it can be grown to high densities on inexpensive substrates, and fermentation scale-up is straightforward (Georgiou, 1988).
However, one of the major problems associated with the expression of heterologous polypeptides in Escherichia coli and related bacteria is the degradation of cloned gene products by host-specific proteases (Baneyx and Georgiou, 1992). In a manner similar to that occurring in eukaryotic cells, energy-dependent processes are important for the degradation of E. coli proteins with abnormal conformations (Goldberg and St. John, 1976; Hershko and Ciechanover, 1982). Most E. coli proteases hydrolyze peptide bonds via an energy-independent pathway. At least 25 proteases and peptidases have been identified in different cellular compartments of E. coli (Lazdunski, 1989; Miller, 1987). The biochemical characterization of these enzymes is incomplete and there is relatively little information on their physiological role. One or more of these proteases may act upon any given polypeptide to effect degradation and thereby reduce yields, sometimes quite drastically.
One approach to solving the problem of low polypeptide production in bacterial host cells has been the use of an inducible expression system in combination with a constitutively protease-deficient bacterial host strain. This method increases polypeptide yields only if the expressed polypeptide is a substrate for the deficient protease. For example, production of an immunologically functional antibody fragment in a constitutively lon.sup.- and/or htpR.sup.- E. coli strain has produced low yields (Field, et al., 1989) even though such strains are protease deficient.
Several strains of E. coli deficient in proteases or genes controlling the regulation of proteases are known (Beckwith and Strauch, 1988; Chaudhury and Smith, 1984; Elish, et al., 1988; Baneyx and Georgiou, 1992). Some of these strains have been used in attempts to efficiently produce proteolytically sensitive polypeptides, particularly those of potential medical or other commercial interest. However, while increased yields of some expressed proteins have been improved, problems of relatively low yield and/or poor growth of the host cell continue to persist.
Singly protease-deficient mutants of Escherichia coli have been reported. These include genetically engineered strains deficient in degP and a spontaneous mutant, UT4400, which lacks the entire ompT gene together with a sizable piece of adjacent DNA (McIntosh, et al., 1979). Mutants carrying large deletions in the ptr gene have been isolated (Chaudhury and Smith, 1984). However, in those bacteria all or at least part of the adjacent genes recC, recB and recD is missing. Such bacteria tend to be unstable and generally exhibit growth defects and low protein production. The recC, recB and recD genes are known to be important for cell viability and stable propagation of plasmids in bacteria such as E. coli. Significant portions of these adjacent genes appear to be missing in the reported ptr deficient mutants.
A ptr mutant strain has also been isolated after chemical mutagenesis (Cheng and Zipster, 1979). Cheng et al. exposed an E. coli culture to the mutagen nitrosoguanidine and by brute force screening isolated three mutants that exhibited decreased rates of degradation of the "auto .alpha." fragment of .beta.-galactosidase which is a substrate for protease III. In vitro, the three point mutants isolated by Cheng et al., exhibited about 5% of the activity detected in wild type strains. However, the three mutants did not affect the degradation of several .beta.-galactosidase nonsense mutants in vivo. This finding led Cheng et al. to conclude that the activity of protease III in vivo in these mutants was probably not affected. If the activity of protease III in vivo is unaffected by the mutations then, obviously, these strains are not suitable for protein expression. Furthermore, Cheng et al. did not investigate whether the mutations they isolated have any deleterious effects on cell growth and plasmid stability.
As found in later studies (e.g., Claverie-Martin et al. 1987), the ptr gene which encodes protease III overlaps the recC genes which encode two exonuclease V subunits. Exonuclease V is an important enzyme for DNA repair, resistance to UV irradiation and the stable maintenance of expression vectors in the cell (Baneyx and Georgiou 1991). Randomly generated mutations in the ptr gene are expected to interfere with the expression of the overlapping recB and recC genes. Mutations that affect the expression of the RecB and RecC subunits of exonuclease V can impair cell viability and substantially decrease the expression of heterologous proteins.
Other singly protease deficient mutants have been produced. Using a genetic engineering approach, Escherichia coli has been mutagenized to produce a cell with a defective periplasmic protease (Beckwith and Strauch, 1988). A degP deletion mutant was constructed and recombined into an E. coli chromosome (Strauch and Beckwith, 1988). Protein A-.beta.-lactamase, a proteolytically sensitive protein, is stabilized three-fold in such a degP mutant (Baneyx and Georgiou, 1989; Baneyx and Georgiou, 1990).
Hara et al. (1991) have isolated transposition mutations that disable another secreted protease, Prc. This protease was first isolated on the basis of its ability to cleave the periplasmic protein Penicillin-Binding Protein 3. Subsequently, it was also identified as a protease that selectively degrades proteins with a non-polar C-terminus and was renamed Tsp (Silber et al. 1992). Prc(tsp) mutant strains exhibit certain abnormal growth characteristics such as inability to form colonies at elevated temperatures when grown in LB media without salt. Additionally, they leak periplasmic enzymes into the growth medium and grow as elongated filaments. Hara et al. (1991) and Silber et al. (1992) did not investigate whether prc(tsp) mutants increase the stability of recombinant secreted proteins or whether they impair cell viability when combined with other loci that impair protein turnover in the cell envelope.
Most proteins are degraded by more than one protease. Therefore, use of mutants deficient in the synthesis of a single enzyme may only partially prevent the degradation of the product. Inactivation of multiple proteolytic enzymes may lead to higher production. The challenge is complex because there is no assurance that disablement or deletion of any given protease or combination of proteases will result in a viable host cell. Additionally, even where singly protease-deficient microorganisms are available, growth and/or viability is typically affected so that yields of overexpressed polypeptide products are low, making such microorganisms of little commercial value.
Heat shock proteins are among the proteolytic enzymes involved in protein catabolism in the cytoplasm. Heat shock protein synthesis is induced by growth at elevated temperatures or by exposure to stressful conditions such as ethanol. Heat shock results in the overproduction of heat shock proteins and is accompanied by increased degradation of puromycyl polypeptide fragment (Strauss et al. 1988). In turn, the accumulation of abnormal polypeptides, resulting from treatment with puromycin, incorporation of canavanine or overproduction of foreign proteins, causes induction of the heat shock response (Goff and Goldberg 1985).
Transcription of the heat shock regulon is controlled by the RNA polymerase sigma factor, .sigma..sup.32. The effect of .sigma..sup.32 mutations on the expression of secreted proteins is not known. There are no secreted proteases whose synthesis is dependent on .sigma..sup.32.
There is therefore a need to provide microorganisms that show good viability, stability and, importantly, are suitable hosts for expression of proteolytically sensitive polypeptides.